The RIO trial: rationale, design, and the role of community involvement in a randomised placebo-controlled trial of antiretroviral therapy plus dual long-acting HIV-specific broadly neutralising antibodies (bNAbs) in participants diagnosed with recent HIV infection-study protocol for a two-stage randomised phase II trial.

BACKGROUND: Antiretroviral therapy (ART) has led to dramatic improvements in survival for people living with HIV, but is unable to cure infection, or induce viral control off therapy. Designing intervention trials with novel agents with the potential to confer a period of HIV remission without ART remains a key scientific and community goal. We detail the rationale, design, and outcomes of a randomised, placebo-controlled trial of two HIV-specific long-acting broadly neutralising antibodies (bNAbs): 3BNC117-LS and 10-1074-LS, which target CD4 binding site and V3 loop respectively, on post-treatment viral control. METHODS: RIO is a randomised, placebo-controlled, double-blinded prospective phase II study. Eligible individuals will have started ART within 3 months of primary HIV infection and have viral sequences that appear to be sensitive to both bNAbs. It will randomise 72 eligible participants 1:1 to the following arms via a two-stage design. In Stage 1, arm A participants are given dual long-acting (LS-variants) bNAbs infusions, followed by intensively monitored Analytical Treatment Interruption (ATI) (n = 36); in arm B, participants receive placebo infusions followed by ATI. The primary endpoint will be time to viral rebound within 36 weeks after ATI. Upon viral rebound, the participant and researcher are unblinded. Participants in arm A recommence ART and complete the study. Participants in arm B are invited to restart ART and enroll into Stage 2 where they will receive open-label LS bNAbs, followed by a second ATI 24 weeks after. Secondary and exploratory endpoints include adverse events, time to undetectable viraemia after restarting ART, immunological markers, HIV proviral DNA, serum bNAb concentrations in blood, bNAb resistance at viral rebound, and quality of life measures. DISCUSSION: The two-stage design was determined in collaboration with community involvement. This design allows all participants the option to receive bNAbs. It also tests the hypothesis that bNAbs may drive sustained HIV control beyond the duration of detectable bNAb concentrations. Community representatives were involved at all stages. This included the two-stage design, discussion on the criteria to restart ART, frequency of monitoring visits off ART, and reducing the risk of onward transmission to HIV-negative partners. It also included responding to the challenges of COVID-19. TRIAL REGISTRATION: The protocol is registered on Clinical. TRIALS: gov and EudraCT and has approval from UK Ethics and MHRA.

Viral inhibition assay: a CD8 T cell neutralization assay for use in clinical trials of HIV-1 vaccine candidates.

We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.

Broad HIV-1 inhibition in vitro by vaccine-elicited CD8(+) T cells in African adults.

We are developing a pan-clade HIV-1 T-cell vaccine HIVconsv, which could complement Env vaccines for prophylaxis and be a key to HIV cure. Our strategy focuses vaccine-elicited effector T-cells on functionally and structurally conserved regions (not full-length proteins and not only epitopes) of the HIV-1 proteome, which are common to most global variants and which, if mutated, cause a replicative fitness loss. Our first clinical trial in low risk HIV-1-negative adults in Oxford demonstrated the principle that naturally mostly subdominant epitopes, when taken out of the context of full-length proteins/virus and delivered by potent regimens involving combinations of simian adenovirus and poxvirus modified vaccinia virus Ankara, can induce robust CD8(+) T cells of broad specificities and functions capable of inhibiting in vitro HIV-1 replication. Here and for the first time, we tested this strategy in low risk HIV-1-negative adults in Africa. We showed that the vaccines were well tolerated and induced high frequencies of broadly HIVconsv-specific plurifunctional T cells, which inhibited in vitro viruses from four major clades A, B, C, and D. Because sub-Saharan Africa is globally the region most affected by HIV-1/AIDS, trial HIV-CORE 004 represents an important stage in the path toward efficacy evaluation of this highly rational and promising vaccine strategy.

Broad HIV epitope specificity and viral inhibition induced by multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults.

A correlation between in vivo and in vitro virus control mediated by CD8+ T-cell populations has been demonstrated by CD8 T-cell-mediated inhibition of HIV-1 and SIV replication in vitro in peripheral blood mononuclear cells (PBMCs) from infected humans and non-human primates (NHPs), respectively. Here, the breadth and specificity of T-cell responses induced following vaccination with replication-defective adenovirus serotype 35 (Ad35) vectors containing a fusion protein of Gag, reverse transcriptase (RT), Integrase (Int) and Nef (Ad35-GRIN) and Env (Ad35-ENV), derived from HIV-1 subtype A isolates, was assessed in 25 individuals. The vaccine induced responses to a median of 4 epitopes per vaccinee. We correlated the CD8 responses to conserved vs. variable regions with the ability to inhibit a panel of 7 HIV-1 isolates representing multiple clades in a virus inhibition assay (VIA). The results indicate that targeting immunodominant responses to highly conserved regions of the HIV-1 proteome may result in an increased ability to inhibit multiple clades of HIV-1 in vitro. The data further validate the use of the VIA to screen and select future HIV vaccine candidates. Moreover, our data suggest that future T cell-focused vaccine design should aim to induce immunodominant responses to highly conserved regions of the virus.

A DNA-based candidate HIV vaccine delivered via in vivo electroporation induces CD4 responses toward the α4ß7-binding V2 loop of HIV gp120 in healthy volunteers.

Administration of a clade C/B' candidate HIV-1 DNA vaccine, ADVAX, by in vivo electroporation (EP) was safe and more immunogenic than intramuscular administration without EP. The breadth and specificity of T-cell responses to full-length Env were mapped. Responses to multiple Env regions were induced, with most focusing on V3/C4 and V2 regions, including the α4ß7 integrin-binding domain. The breadth of responses induced by this DNA vaccine regimen was comparable to that of viral-vectored vaccine regimens.

Measuring human T cell responses in blood and gut samples using qualified methods suitable for evaluation of HIV vaccine candidates in clinical trials.

The next generation of candidate HIV vaccines include replicating vectors selected for tropism to mucosal sites, where an efficacious T cell response will be required to limit T cell replication and HIV associated CD4 T cell loss. To fully assess immunogenicity of such candidates, there is a need to develop robust quality controlled analysis of gut derived HIV specific CD8+ T-cell responses. Despite obvious challenges in obtaining sufficient amounts of tissue, the highly compartmentalised nature of the mucosal immune responses, requires the assessment of CD8 T cells isolated directly from local tissue before any conclusions regarding the induction of mucosal responses are made. Here we describe the optimisation and subsequent qualification of a qualitative and quantitative polychromatic flow cytometry assay to assess antigen specific CD8+ T cells isolated from the gut, using samples from HIV positive and negative volunteers. Internal quality controls monitored over time, combined with the use of quality gating and standard operating procedures were used to demonstrate the generation of robust and reliable data.

Performance of two commercially available sequence-based HIV-1 genotyping systems for the detection of drug resistance against HIV type 1 group M subtypes.

The use of genotyping assays for the detection and evaluation of drug resistance mutations within the polymerase gene of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly relevant in the clinical management of HIV-1 infection. However, genotypic resistance assays available currently have been optimised for genetic subtype B strains of the virus and many clinical centres are presented with strains from subtypes A, C, and D. In the present report, we compare the performance of two sequence-based commercially available kits, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA) and the TruGene HIV-1 Genotyping Kit (Visible Genetics, Toronto, Ontario) against a panel of 35 virus isolates from HIV-1 Group M (subtypes A-J). Full-length consensus sequences were generated by the ViroSeq genotyping system for 26 of 31 (83.8%) of the isolates tested, in contrast to the TruGene genotyping system, which generated 16 of 30 (53%) usable sequences overall. Overall, subtype B isolates were sequenced with a greater degree of success than non-subtype B isolates. Discrepancies were found between the consensus sequences reported by each system for each sample (mean difference 1.0%; range 0.0-3.2%), but these appeared to be random and did not affect interpretation of the major resistance codons. In addition, both systems were able to amplify template RNA from low copy viral load plasma samples (10(2)-10(3) RNA copies/ml) taken from a random selection of patient samples encompassing subtypes A-C. While the availability of these genotyping systems should facilitate studies of HIV-1 drug resistance in countries in which these subtypes are prevalent, the performance against subtypes other than B needs to be improved.